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Development of ISSR markers for a Bulgarian tomato breeding collection aiming to improve antioxidant compounds in fruits

Authors:
  • Center of Plant Systems Biology and Biotechnology
  • University of Food Technologies; Bulgarian Academy of Sciences

Abstract and Figures

The Inter-Simple Sequence Repeat marker technique was applied to a set of tomato genotypes that were found to diverge in antioxidant compounds in fruits. As marker-assisted selection is becoming a tool that brings major advantages for breeding programs need is rising to use it to quickly respond to consumer demand. Being part of a system for efficient plant breeding in many crops the aim of the present study was to test Inter-Simple Sequence Repeats as candidate tool for incorporating in a breeding program aiming to improve antioxidant properties of tomato fruits. Application of Inter-Simple Sequence Repeat markers resulted in obtaining adequate representation of every accession from the selected group. This opens the opportunity for using the system in confirming the hybrid nature of the F1 progenies and further accelerating the breeding of genotypes for use as healthy food. © 2017, National Centre for Agrarian Sciences. All rights reserved.
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405
Bulgarian Journal of Agricultural Science, 23 (No 3) 2017, 405–410
Agricultural Academy
DEVELOPMENT OF ISSR MARKERS FOR A BULGARIAN TOMATO BREEDING
COLLECTION AIMING TO IMPROVE ANTIOXIDANT COMPOUNDS IN FRUITS
ANGELOV MICHAIL1; BILYANA IVANOVA1; ATANAS PAVLOV2; DANIELA GANEVA3;
ZHIVKO DANAILOV4; BOJIN BOJINOV*1
1 Agrucultural University of Plovdiv, Department of Genetics and Plant Breeding, BG-4000 Plovdiv
2
Bulgarian Academy of Science, The Stephan Angeloff Institute of Microbiology, Laboratory of Applied Biotech-
nologies, BG-4000 Plovdiv, Bulgaria
3 Agrucultural Academy, Maritsa Vegetable Crops Research Institute, BG-4000 Plovdiv, Bulgaria
4 Bulgarian Academy of Science, Institute of Plant Physiology and Genetics, Bldg. 21, BG-1113 Sofi a, Bulgaria
Abstract
Angelov, M., B. Ivanova, A. Pavlov, D. Ganeva, Zh. P. Danailov and B. M. Bojinov, 2017, Development of ISSR
markers for a Bulgarian tomato breeding collection aiming to improve antioxidant compounds in fruits. Bulg. J.
Agric. Sci., 23 (3): 405–410
The Inter-Simple Sequence Repeat marker technique was applied to a set of tomato genotypes that were found to diverge
in antioxidant compounds in fruits. As marker-assisted selection is becoming a tool that brings major advantages for breeding
programs need is rising to use it to quickly respond to consumer demand. Being part of a system for effi cient plant breeding
in many crops the aim of the present study was to test Inter-Simple Sequence Repeats as candidate tool for incorporating in
a breeding program aiming to improve antioxidant properties of tomato fruits. Application of Inter-Simple Sequence Repeat
markers resulted in obtaining adequate representation of every accession from the selected group. This opens the opportunity
for using the system in confi rming the hybrid nature of the F1 progenies and further accelerating the breeding of genotypes for
use as healthy food.
Key words: Solanum lycopersicum; molecular markers; Inter-Simple Sequence Repeats; ISSRs
*Corresponding author: bojinov@au-plovdiv.bg
Introduction
Genotype identifi cation is of great importance for seed
production and cultivar propagation. In the recent years
marker-assisted selection (MAS) is becoming a tool that
brings major advantages through allowing for identifi cation
of genotypes based on the unique sequences in their genome
and following these sequences through the progenies of in-
tercrossed species.
The potential of the molecular techniques for develop-
ing “DNA passports” for tomato cultivars and hybrids is dis-
cussed in a number of publications (Kochieva et al., 2002a;
Kochieva et al., 2002b; Suliman-Pollatschek et al., 2002;
Cooke et al., 2003; He et al., 2003; Tikunov et al., 2003; Park
et al., 2004; Frary et al., 2005; García-Martínez et al., 2006).
In many of these reports a tendency for decreasing of the
genetic diversity in modern cultivars is discussed, the pro-
cess being particularly notable in the recent years and within
the breeding programs for hybrid production (Archak et al.,
2002). Testing of 27 cultivars widely grown in India with
RAPDs (Archak et al., 2002) demonstrated that the variabil-
ity within the primitive local and older introduced cultivars
is higher than that within the recently developed genotypes.
The overall variability in the studied genotypes was rather
406 Angelov Michail, Bilyana Ivanova, Atanas Pavlov, Daniela Ganeva, Zhivko Danailov, Bojin Bojinov
low in spite of the presence of cultivars from both primary
and secondary centers of origin of the crop.
RAPDs were applied by Kochieva et al. (2002b) for the
analysis of a collection composed of 43 accessions and 10
cultivars from many representatives of the Solanum lycoper-
sicum species. The applied 8 RAPD primers allowed group-
ing of the genotypes according to the known phylogenetic
relations. However even after producing 248 polymorphic
bands within this group of genotypes obtaining a unique pat-
tern for each of them could not be achieved. The same group
(Kochieva et al., 2002а) attempted the application of Inter-
Simple Sequence Repeat markers (ISSRs) where the combi-
nation of 14 ISSR primers for characterizing 54 genotypes
resulted in 304 polymorphic fragments and this extended set
was able to produce unique pattern for each of the tested ac-
cessions.
ISSR marker system was used by Tikunov et al. (2003)
for comparative characterization of Lycopersicon escul-
entum, Lycopersicon pennellii, Lycopersicon cheesmanii,
Lycopersicon humboldtii, Lycopersicon hirsutum, together
with two isogenic lines from Lycopersicon esculentum.
In this study 9 of the 14 primers used were able to inde-
pendently differentiate all the species. Authors concluded
that in addition to their high reliability, reproducibility
and speed of development ISSRs can identify signifi cant
number of polymorphisms in tomato, which characteristics
are extremely important for the practical applications of
a DNA marker system in plant breeding. Similarly ISSR
markers were tested on 10 genotypes to demonstrate their
relative reproducibility as compared to SSR and RAPD
markers (Sanghani and Mandavia, 2013). The same system
was used by Henareh et al. (2016) to assess genetic diver-
sity in tomato landraces from Turkey. Furthermore ISSR
markers were demonstrated to bare potential in associating
with agronomically important traits such as hybrid perfor-
mance (Hernandez-Ibanez et al., 2014) and drought toler-
ance (Metwali et al., 2016).
In 2003 Cooke et al. studied the applicability of the SSR
markers to distinguishability, uniformity and stability (DUS)
testing in accordance with the UPOV standards. Analyzing
36 plants from 10 widely used cultivars demonstrated that 9
of the 10 cultivars were homogenic in the 6 loci tested thus
meeting the UPOV standards. In spite of fully correspond-
ing to the phenotypic descriptors of the organization 3 of the
plants from the last cultivar carried a different allele in one
of the tested loci. Current DUS testing standard for tomatoes
allows for maximum 1 in 20 plants deviating for some of the
characteristics, thus this cultivar would not pass the test if the
results from the DNA analyses were equally acceptable to
the phenotypic characteristics. Similar or even higher levels
of heterogeneity were observed by Bredemeijer et al. (2002)
when constructing the European tomato database. Testing of
500 cultivars in that study resulted in 30% of them express-
ing some heterogeneity.
The analysis of the available literature indicates that the
lack of preliminary information about the Bulgarian geno-
types suggests that the initial screening of such collection
should be made with ISSR primers. Due to their multilocus
assessment capability in a single reaction and the relatively
uniform distribution within tomato genome (Tikunov et al.,
2003) their application bares suffi cient potential of identify-
ing unique profi les for each genotype.
The aim of the present report is to present the results
from testing ISSRs as candidate technique for identifi cation
of diversity in tomato genotypes within one Bulgarian breed-
ing collection, assembled with the aim of improving the anti-
oxidant content of cultivated varieties.
Materials and Methods
Plant material
Breeding lines 975, 984, 53β, 1116, 1140 as well as va-
rieties IZK Alia and Plovdivska karotina were used in the
present study. All cultivars and breeding lines belong to the
Solanum lycopersicum species. Accessions are maintained at
“Maritsa” Vegetable Crops Research Institute and are used in
establishing a breeding program as donors of antioxidative
stress ingredients (ascorbic acid, lycopene, beta-carotene,
anthocyanins, etc.) and other fruit quality traits (ex. dry mat-
ter content). Furthermore, some of these lines proved valu-
able sources for traits, related to improving agronomic per-
formance as crops.
The accessions used in the present study were homog-
enized and stabilized for the main approbation traits of the
fruits and plants and controlled for the main biochemical
compounds of the fruits. Only the plants fully corresponding
to the phenotypic descriptors of the UPOV for the cultivar/
line were used for DNA extraction. These genotypes were
never before tested for their homogeneity with the use of
molecular markers.
ISSR analysis
ISSR analysis was performed by testing a number of
primers that demonstrated high reproducibility and poly-
morphism identifi cation in our previous studies (Bojinov and
Danailov, 2009). The selection of the fi nal group of 7 prim-
ers (Table 1) was based on our previous experience and the
screening of the selected primers against the available data
on the frequency of tandem repeats in tomato genome.
407
Development of ISSR Markers for a Bulgarian Tomato Breeding Collection Aiming to Improve Antioxidant...
PCR reactions were performed in 25 ul volume with the
following cycling regime: denaturing at 94°C for 3 min, 40
cycles of 94°C – 1 min, AT – 45 sec, 72°C – 45 sec, fol-
lowed by fi nal extension of 72°C – 4 min, where AT is the
annealing temperature for each primer calculated according
to Kochieva et al. (2002a).
PCR products were analyzed through separation in 2%
agarose gels and staining with ethidium bromide.
Results and Discussion
DNA extraction procedure resulted in obtaining high
quality genomic DNA from all accessions (Figure 1). Be-
tween 300 and 500 μg of genomic DNA was recovered in
most of the cases. When the quantity or quality of the ob-
tained DNA was considered unsatisfactory the extraction
was repeated, so that the above criteria can be met.
Due to reasons discussed above our system for screen-
ing was based on Inter-Simple Sequence Repeat markers
(ISSRs). We screened individual plants from each of the
accessions (Figure 2) to verify the capacity of the chosen
marker system to reveal suffi cient number of polymorphisms
within each of them. As Figure 2 demonstrates some of the
ISSR primers produced few polymorphic bands, which were
not suffi cient to identify every plant from the accession. This
was expected as tomatoes are self-pollinating crop and thus
not much heterogeneity is expected within each genotype.
The results from this initial screening demonstrated however
the capacity of the selected marker system to differentiate even
individual plants within the accessions. The fi gure represents
the results obtained with 7 plants from one of the genotypes.
The above results were obtained by using genomic DNA
extracted from single plants for each of the accessions. To
verify the usability of individual bands as specifi c for any
genotype bulked DNA samples should be preferably used.
However the use of bulked DNA samples obscures the levels
of heterogeneity within each genotype and thus can jeopar-
dize the reproducibility of the results if heterogenic material
is included in the sample. The chances of such heterogeneity
occurring in each sample are not negligible as discussed by
Cooke et al. (2003) and Bredemeijer et al. (2002). With no
preliminary DNA data on the genotypes used in our study
this possibility had to be examined.
Table 1
Description of the tested ISSR primers
Prim er DNA sequence Length (bp)
ISSR 1 (AG)8C+TC 19
ISSR 2 (AG)8C+TG 19
ISSR 3 (GA)8T17
ISSR 4 (GA)8C17
ISSR 5 (GA)8YC 18
ISSR 6 (AG)8YT 18
ISSR 7 (GT)8YC 18
Fig. 1. Results from extracting tomato genomic DNA
from young leaves of selected genotypes. Each lane is
loaded with 10 ul of the fi nal solution of extracted DNA
and stained with ethidium bromide
lanes 1-7 – individual plants from Line 984,
lanes 8-14 – individual plants from Line 975,
lanes 15-16 – individual plants from line 1140
Fig. 2. Revealing heterogeneity within accessions
with primer ISSR2
lane 1 – standard sized DNA with bands of 500 and 1000 bp
appearing as strong signals, lanes 2-8 – products from PCR
reactions with individual plants of line 21β, lane 9 – control
408 Angelov Michail, Bilyana Ivanova, Atanas Pavlov, Daniela Ganeva, Zhivko Danailov, Bojin Bojinov
The evaluation of the existing heterogeneity within each of
the cultivated genotypes was performed through testing each
of the selected 7 plants per accession with all ISSR primers
and looking for appearance of uncommon bands. Interest-
ingly none of the genotypes showed complete uniformity of
the profi les of individual plants (as would be expected from
strictly self-pollinating species). In all of the genotypes at least
one plant demonstrated differences in the obtained profi les. As
demonstrated on Figure 3 plant No. 4 (Lane 5) had the band at
about 380 bp missing which band is present in all other plants
from the same accession. On the other hand a band at 410 bp
appears as relatively strong signal in that plant which is un-
common to other representatives of the same accession.
As expected testing 7 ISSR primers resulted in revealing
different levels of polymorphism within accessions. How-
ever the use of the 3 ISSR primers that together produced
most polymorphic bands (55 in total) resulted in obtaining
clustering of the tested set of plants that corresponds well to
their initial designation (Figure 4). In general the grouping
of the plants corresponds to their designated accession with
occasional individuals appearing moved to a closely related
cluster. Notable exceptions are the accessions “IZK Alia”,
Line 1116, and Line 984 where plants were separated in two
distinguished groups. These exceptions would have inspired
doubts in the applicability of our system if they were not
corresponding to the metabolite profi les of the same plants
(our unpublished data). However we have found these no-
Fig. 3. Revealing heterogeneity within accessions with
primer ISSR 1
lane 1 – standard sized DNA with bands of 500 and 1000 bp
appearing as strong signals, lanes 2-8 – PCR products
from individual plants of line 1140, lane 9 – control
Fig. 4. Dendrogram of genotype distribution based
on 55 polymorphic bands produced by three ISSR
primers
Dendrogram using Ward Linkage
Rescaled Distance Cluster Combine
409
Development of ISSR Markers for a Bulgarian Tomato Breeding Collection Aiming to Improve Antioxidant...
table exceptions in DNA profi les to correspond well to the
deviations in metabolite profi les (antioxidant compounds in
fruits) of the same plants.
The observed differences between the plants in each va-
riety that appeared identical in their phenotypic character-
istics at the time of initial selection can be attributed to a
number of reasons. One possible explanation of the observed
disagreement between the phenotypic homogeneity and the
identifi ed genotypic heterogeneity is that the heterozygous
loci are unrelated to the traits the genotypes are selected for
during the breeding process. On the other hand Bredemei-
jer et al. (2002) suggested that such results can be explained
by the presence of residual heterogeneity mainly in the non-
coding sequences and/or within the quantitative trait loci
(QTLs) with very small phenotypic effect. Eliminating such
residual heterogeneity would be impossible with the classi-
cal breeding approaches.
It is questionable whether attempting to eliminate any
such heterogeneity with the use of molecular markers is of
any practical use as in most cases they could not be associ-
ated with any phenotypic effects. Whether in the cases of
differing plants from Line 1140, Line 984 and IZK Alia the
identifi ed marker diversity could be linked to the observed
variation in metabolite profi les is a matter of further studies.
The demonstrated capacity of the ISSRs to identify the
presence of such heterogeneity in the studied material pro-
vides striking evidence in support of the careful selection of
the marker system. The marker system of choice that could
be used in the breeding programs has to be reliable, effi cient,
fast and easy to implement. In cases where a single plant
deviates from the group of 7 of the same genotype discard-
ing that plant can easily result in eliminating the heterogene-
ity from the selected genotype. The other 6 plants can be
safely used to produce pure material for seed multiplication
of that genotype. However in cases where more than one
plant appears with a different profi le (the cases in Figure 2
and Figure 3) the selection of the representative set of bands
for a particular genotype might be diffi cult, especially when
the plants appear divided into two more or less equal groups
(like was the case with cv. Plovdivska karotina, data not pre-
sented). In such cases the question what should be consid-
ered “signifi cant deviation” arises and whether a representa-
tive profi le for that particular variety can be produced so that
it can be distinguished from other varieties.
Conclusions
The results presented in this paper demonstrate that ISSR
markers can effi ciently differentiate tomato genotypes even
within phenotypically highly homogenous accessions. Ap-
plication of ISSRs revealed not only suffi cient polymor-
phisms between different accessions, but was capable of
identifying deviating plants within individual accessions.
This demonstrates the power of the proposed technique to
resolve differences even within presumably highly homog-
enous genotypes like the self-pollinating cultivars from So-
lanum lycopersicum species. As evidenced in obtained clus-
tering (Figure 4) the ISSR markers are capable of not just
identifying diversity in the studied accessions, but are appar-
ently baring the potential to effi ciently identify individuals
with differing metabolite profi les within varieties. The ease
of use and the high reliability of the system make it very
good candidate for practical application in tomato breeding
programs.
Acknowledgements
The present study was supported through a Research
Grant B01/16-2012 from the Bulgarian National Science
Fund.
References
Archak, S., J. L. Karihaloo and A. Jain, 2002. RAPD markers
reveal narrowing genetic base of Indian tomato cultivars. Cur-
rent Science, 82 (9): 1139-1143.
Bojinov, B. M. and Zh. P. Danailov, 2009. Applicability of ISSRs
for Genotype Identifi cation in a Tomato Breeding Collection.
Acta Horticulturae, 830: 63-70.
Bredemeijer, G. M. M., R. J. Cooke, M. W. Ganal, R. Peeters,
P. Isaac and Y. Noordijk, 2002. Construction and testing of a
microsatellite database containing more than 500 tomato variet-
ies. Theoretical and Applied Genetics, 105: 1019-1026.
Cooke, R. J., G. M. M. Bredemeijer, M. W. Ganal, R. Peeters,
P. Isaac, S. Rendell, J. Jackson, M. S. Röder, V. Korzun, K.
Wendehake, T. Areshchenkova, M. Dijcks, D. Laborie, L.
Bertrand and B. Vosman, 2003. Assessment of the uniformity
of wheat and tomato varieties at DNA microsatellite loci. Eu-
phytica, 132: 331-341.
Frary, A., Y. Xu, J. Liu, S. Mitchell, E. Tedeschi and S. Tanks-
ley, 2005. Development of a set of PCR-based anchor markers
encompassing the tomato genome and evaluation of their use-
fulness for genetics and breeding experiments. Theoretical and
Applied Genetics, 111: 291-312.
García-Martínez, S., L. Andreani, M. Garcia-Gusano, F. Geuna
and J. J. Ruiz, 2006. Evaluation of amplifi ed fragment length
polymorphism and simple sequence repeats for tomato germ-
plasm fi ngerprinting: utility for grouping closely related tradi-
tional cultivars. Genome, 49: 648-656.
He, C., V. Poysa and K. Yu, 2003. Development and characteriza-
tion of simple sequence repeat (SSR) markers and their use in
determining relationships among Lycopersicon esculentum cul-
tivars. Theoretical and Applied Genetics, 106: 363-373.
Henareh, M., A. Dursun, B. Abdollahi-Mandoulakani and K.
410 Angelov Michail, Bilyana Ivanova, Atanas Pavlov, Daniela Ganeva, Zhivko Danailov, Bojin Bojinov
Haliloğlu, 2016. Assessment of genetic diversity in tomato
landraces using ISSR markers. Genetika, 48 (1): 25-35.
Hernandez-Ibanez, L., J. Sahagun-Castellanos, J. E. Rodri-
guez-Perez, M. G. Pena-Ortega, and L. M. Rodriguez-
Martinez, 2014. Prediction of tomato hybrid performance with
genomic markers. PHYTON-International Journal of Experi-
mental Botany, 83: 311-318.
Kochieva, E. Z., N. N. Ryzhova, I. A. Khrapalova and V. A.
Pukhalskyi, 2002a. Genetic diversity and phylogenetic rela-
tionships in the genus Lycopersicon (Tourn.) Mill. as revealed
by Inter-Simple Sequence Repeat (ISSR) analysis. Russian
Journal of Genetics, 38 (8): 958-966.
Kochieva, E. Z., N. N. Ryzhova, I. A. Khrapalova and V. A.
Pukhalskyi, 2002b. Using RAPD for estimating genetic poly-
morphism in and phylogenetic relationships among species of
the genus Lycopersicon (Tourn.) Mill. Russian Journal of Ge-
netics, 38 (9):1104-1108.
Metwali, E. M. R., R. Carle, R. M. Schweiggert, N. M. Kadasa
and O. A. Almaghrabi, 2016. Genetic diversity analysis based
on molecular marker and quantitative traits of the response of
different tomato (Lycopersicon esculentum Mill.) cultivars to
drought stress. Archives of Biological Sciences, 68 (2): 427-
438.
Park, Y. H., M. A. West and D. A. St Clair, 2004. Evaluation of
AFLPs for germplasm fi ngerprinting and assessment of genetic
diversity in cultivars of tomato (Lycopersicon esculentum L.).
Genome, 47: 510-518.
Sanghani, A. and M. Mandavia, 2013. Characterization of tomato
(Lycopersicon lycopersicum Mill.) genotypes through RAPD,
ISSR and SSR markers. Indian Journal of Agricultural Bio-
chemistry, 26 (2): 141-147.
Suliman-Pollatschek, S., K. Kashkush, H. Shats, J. Hillel and U.
Lavi, 2002. Generation and mapping of AFLP, SSRs and SNPs
in Lycopersicon esculentum. Cellular & molecular biology let-
ters, 7: 583-597.
Tikunov, Yu. M., L. I. Khrustaleva and G. I. Karlov, 2003. Ap-
plication of ISSR markers in the genus Lycopersicon. Euphyt-
ica, 131: 71-80.
Received December, 23, 2016; accepted for printing March, 10, 2017
... The same markers were used to evaluate tomato genotypes with different antioxidant content. The obtained fingerprints were used to confirm the nature of the hybrids in breeding programs, thus accelerating the selection process [108]. ...
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The level of polymorphism in tomato was studied using ISSR-PCR. Five tomato species: Lycopersicon esculentum, Lycopersicon pennellii, Lycopersicon cheesmanii, Lycopersicon humboldtii, Lycopersicon hirsutum and two Lycopersicon esculentum substitution lines IL 6-3 and WSL 6 were analyzed. ISSR-PCR was performed with fourteen primers. Nine of these fourteen primers were individually able to distinguish all tomato species. The data were used to create a phylogenetic tree of the five tomato species. The tree showed complete correspondence to previous phylogenetic investigations. ISSR-PCR on two Lycopersicon esculentum substitution lines IL 6-3 and WSL 6 enabled us to place thirteen ISSR markers on the classical map of Lycopersicon esculentum chromosome 6. Some of the markers were not located in the pericentromeric region. Using one ISSR and one RGA (Resistance-gene analogs) primer resulted in fingerprints having some new bands compared with ISSR fingerprints.
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Molecular characterization of ten tomato genotypes was carried out using RAPD, ISSR and SSR markers. The RAPD data showed that primer OPK-03 was the best primer resulting good amplification with maximum PIC value (0.842). Dendrogram constructed using the RAPD data clearly distinguished all genotypes. It was revealed that the genotypes NDT-9 and HADT- 145 found in one cluster shared maximum similarity (82.1%); however, genotype JTL-04-108 was out grouped from other nine genotypes and shared minimum similarity (50.7%). The ISSR data showed that primer K-13 was the best primer which exhibited good amplification with maximum PIC value (0.858). Jaccard’s similarity coefficient ranged from 0.507 to 0.821. The ISSR results indicated that maximum similarity of 82.1% was found between NDT-9 and HADT-145, while minimum similarity of 50.7% was obtained between GT-1 and JTL-04-108. From the SSR data it was found that the primer LETTC 002 was the best primer showing good amplification with maximum PIC value (0.749). Jaccard’s similarity coefficient ranged from 0.519 to 0.846 and revealed that HADT-145 and NDT-9 showed maximum variability compared to other eight genotypes. The combined RAPD, ISSR and SSR analysis revealed that out of ten genotypes HADT-145 and NDT-9 showed maximum similarity (73.7%). The lowest similarity of 58.1% was found between GT-1 and JTL-04-108. © 2013, Indian Society of Agricultural Biochemists. All rights reserved.
Article
RAPD genome analysis of 53 species and cultivars of the genusLycopersicon (Tourn.) Mill. revealed their high genetic polymorphism (Tourn.) Mill., based on which their phylogenetic relationships were inferred. In total, 248 polymorphic DNA fragments were amplified. Intraspecific polymorphism was maximum (79%) in L. peruvianum and minimum (9%) in L. parviflorum. In general, genome divergence among cross-pollinating tomato species was substantially higher than in self-pollinating species. An UPGMA dendrogram constructed from the RAPD patterns was consisted with the Lycopersicon phylogeny inferred from the molecular data of RFLP, ISSR, and microsatellite analyses and with a classification based on morphological characters. The relationships of taxa within the genus Lycopersicon are discussed.
Article
By analysing a number (20–38) of individuals from selected varieties of wheat and tomato, we have been able to assess intra-varietal uniformity at certain micro satellite (simple sequence repeat, SSR) loci. In total, 45 varieties of wheat were analysed at between 7–9 different SSR loci, and 10 varieties of tomato were analysed at six loci. The results showed that there was variation both between varieties and between microsatellites in the degree of non-uniformity observed, and it was possible to identify a number of different probable sources of non-uniformity. Twenty-four of the wheat varieties and nine of the tomato varieties were sufficiently uniform to meet the standards currently applied for distinctness, uniformity and stability (DUS) testing using phenotypic characteristics. The implications for the potential future use of SSRs in DUS testing are discussed.